Journal of Soil Science and Plant Nutrition, 2013, 13(1), 11-21

Effects of soil properties on phosphorus fractions in

subtropical soils of Iran

E. Adhami1 ,H.R. Owliaie1, R. Molavi1, M. Rezaei Rashti2,3*, M. Esfandbod2,3.

1
Soil Science Department, Yasouj University; Yasouj, Iran. 2Griffith School of Environment, Griffith University, Nathan, QLD.,
4111, Australia. 3Environmental Futures Centre, Griffith University, Nathan, QLD., 4111, Australia. Corresponding author:
m.rezaeirashti@griffith.edu.au

Abstract

The distribution of inorganic P fractions and their relationships with soil properties was studied in 17 slightly acidic
to slightly alkaline soils (pH range 5.37 to 7.61). The soils were selected from agricultural fields of the north of Iran.
Inorganic P fractionation included successive extraction with NaOH (NaOH-P), citrate-bicarbonate (CB-P), citrate
two times (C1- and C2-P), Citrate-ascorbate (CAs-P), citrate-dithionite-bicarbonate (CBD-P), sodium-acetate
buffer (NaOAc-P) and HCl (HCl-P). Results showed that the abundance of P fractions was in the order NaOH-P
(35.67 mg kg-1) , NaOAc-P (39 mg kg-1), C2-P (49 mg kg-1), CBD-P (102 mg kg-1), CB-P (136 mg kg-1), CAs-P
(156 mg kg-1), C1-P (197 mg kg-1), HCl-P (417 mg kg-1). Among soil properties, pH had almost a linear negative
relationship with NaOH-P and a linear positive relationship with HCl-P; in addition, it significantly affected C1-P,
CAs-P and NaOAc-P. Oxalate extractable Fe showed a significant positive correlation with NaOH-P and CAs-P,
while calcium carbonate equivalent (CCE) had a significant positive correlation with NaOAc-P and HCl-P.

Keywords: Inorganic phosphorus fractions, agricultural fields, pH.

11
12 Adhami et al.
1. Introduction
Phosphorus (P) fractionation is an applicable technique
to determine the P status of soils and to study the
chemistry and genesis of soils (Chang and Jackson,
1957; Cross and Schlesinger, 1995). Accurately
characterizing P forms is a prerequisite to developing
effective remediation strategies to minimize the adverse
environmental impact of agricultural expansion.
Knowledge of P fractions is important for evaluation its
status in soil and understanding soil chemical properties
that influence soil fertility and environmental quality.
Soil P fractionation has been investigated since 1957
and was later applied to soils and sediments to overcome
the limited information that total P analysis can provide
(Zhou et al., 2001).
Principles of the fractionation methods are to
displace PO4-P from its sorption sites (through anion
competition), to alter the adsorption surface, or to
dissolve compounds containing P. The most easily
desorbed and labile compounds are removed first
with dilute reagents and the strength of the extractants
increases stepwise, in order to separate more strongly
bound P forms. Sequential chemical extraction methods
have often been used to study the nature of P forms
in soils and sediments. These methods are based on
the selective extraction of operationally defined P
fractions by using single extractants in a sequential
manner (Hieltjes and Lijklema, 1980; Olsen and
Sommers, 1982; Jiang and Gu, 1989; Ruiz et al.,
1997). In calcareous soils, P is mainly bound to
adsorption surfaces at low (<10-4.5 M) concentrations
of orthophosphate in solution (Borrero et al., 1988;
Tunesi et al., 1999), whereas it is mainly precipitated
as Ca phosphates at higher concentrations (Castro and
Torrent, 1998; Tunesi et al., 1999). Soils that dominate
humid temperate and tropical regions are highly
weathered, acidic and dominated by large quantities
of sesquioxides. These soils easily adsorb and
geochemicaly fix phosphorus in many cases leading
to phosphorus limitations (Cross and Schlesinger,
1995). In acid to neutral soils, Fe and Al phosphates
are the typical precipitation products (Lindsay, 1979;
Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21
Pierzynski et al., 1990; Cross and Schlesinger, 1995).
However, some heavily fertilized acidic soils contain
significant amounts of Ca phosphates (Lookman et al.,
1996), and hydroxyapatite has been found as a product
of the dissolution of superphosphates in acidic and
slightly acidic soils (Kumar et al., 1994). Pierzynski et
al. (1990) showed that P-rich particles resulting from
fertilizer dissolution in acid soils included not only Al
and Fe but also Si and Ca.
Soils of Guilan province, in the north of Iran, are
characterized by acidic to neutral pH, and more Fe and
Al oxides than soils from other parts of the country
(Ghasemi Fasaie et al., 2007). The climate is subtropical
with an average rainfall of 1000 mm (Bahrami et al.,
2010). Soil characteristics and suitability of the area
for intensive crop production favor high application of
inorganic P fertilizers annually, which has an important
effect on the quality of surface water reservoirs
(Sharpley and Tunney, 2000). Many studies has been
conducted about P chemistry and fractions in other parts
of Iran (Khanmirzaie et al., 2009; Adhami et al; 2007
and 2006) while, such studies are rare in the north of
Iran. The objectives of this study were to evaluate the
status of P fractions in soils from agricultural fields of
Guilan, in the north of Iran, and also investigate the
relationships between soil properties and inorganic P
fractions.
2. Materials and methods
Seventeen surface soil samples (0 - 0.30 m depth) with
a wide range of physical and chemical characteristics
from Guilan province in Iran were selected for this
experiment. The soils were all from agricultural
fields. One composite sample, consisted of at least 8
samples, of each soil was collected. The soil samples
were air-dried and passed through a 2-mm sieve
before analysis. Sand, silt and clay content were
determined by hydrometer (Gee and Bauder, 1986).
Cation exchange capacity (CEC; Sumner and Miller,
1996) by substitution of exchangeable cations with
 Effect of soil properties on phophopus fraction in subtropical soils of Iran 13

sodium acetate, calcium carbonate equivalent (CCE)

by neutralizing with HCl (Loeppert and Suarez,
1996), organic matter content (Nelson and Sommers,
1996) by wet oxidation process, pH from a saturated
paste (Thomas, 1996), and oxalate-extractable Fe (Feo;
Loeppert and Inskeep, 1996) were determined (Table 1).

The inorganic P sequential fractionation scheme

(Table 2) followed the Ruiz et al. (1997) sequence.
Briefly, the procedure includes successive extraction
with NaOH to remove soluble/exchangeable P and
Al- and Fe-bound P; Na citrate bicarbonate (CB-
P) to extract P readsorbed in the previous step and
labile pedogenic Ca-phosphates; Na citrate 2 times
(C1 and C2-P) reagents for extracting pedogenic Ca-
P; sodium-citrate ascorbate (CAs-P) for P occluded
in poorly crystalline Fe oxides; citrate-dithionite
bicarbonate (CBD-P) for P occluded in crystalline Fe
oxides; Na acetate buffer for Ca-P excluding lithogenic
apatite; and HCl to remove mostly lithogenic apatite.
All extractions were carried out on 1-g samples,
in duplicate, with a ratio of 1: 40 soil: extractant.
Following each step the samples were centrifuged at
6000g for 15 min and the supernatant filtered through
Whatman No. 42. Reactive P in the supernatant was
determined using the ascorbic acid method at 882
nm (Murphy and Riley, 1962). Interference with
citrate and dithionite was overcome by enrichment
with ammonium molybdate (Ruiz et al., 1997) and
ammonium persulfate, respectively.

3. Results and discussion

3.1. NaOH-P

The mean of NaOH-P was 35.67 mg kg-1 and it

ranged from 13.2 to 72.61 mg kg-1 which accounted
for 3.16 % of the sum of the fractions. The amount
and percentage of this fraction was obviously higher
than those reported by Adhami et al. (2007) for 16
calcareous soils of the south of Iran. Most of the
fractionation sequences for both calcareous and non-
Figure 1. Relationships between a) NaOH-P and pH;
b) citrate-ascobate extractable P (CAsP) and Oxalate
extractable Fe ( Feo); c) Relationship between HC1-P and
pH

Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21

14 Adhami et al.
calcareous soils starts with NaOH and attribute it to
Fe- and Al-P (Chang and Jackson, 1975; Williams et
al., 1971; Solis and Torrent, 1989; and Ruiz et al.,
1997). This fraction constitutes a small fraction of P
in calcareous soils (Solis and Torrent, 1989; Adhami
et al., 2007). Although Chang and Jackson (1957)
observed that NaOH could dissolve FePO4 2H2O,
its ability to extract Fe- and Al-P in the presence of
CaCO3 has been questioned (Williams et al., 1971). In
the present study, NaOH-P constituted a higher portion
of inorganic P in more acidic soils (6-11 % of the sum
of P fractions) than neutral to slightly basic soil (2-6
% of the sum of P fractions). The content of NaOH-P
was lower than those reported by Ruiz et al. (1997) in
10 neutral to acidic soil (28 to 657 mg kg-1). Ruiz et
al. (1997) reported that in more acidic soils NaOH-P
accounted for a substantial amount of the sum of
inorganic P fractions.
Relationships between NaOH-P and soil properties
showed that it was negatively correlated with CCE
and pH; while it was positively correlated with Feo
(Table 4). Among these properties, pH was the best
variable to describe NaOH-P variation (Figure 1a).
Tyler (2002) studying 110 soils of northeast Sweden
reported a close, almost linear, relationship between
soil acidity and P-Fe. It was observed that P-Fe
fraction constituted about 50% of inorganic P in most
acid soils but less than 1% in moderately alkaline
(calcareous) soils. Relationship between NaOH-P and
soil properties is in agreement with the general assumption
that NaOH extracts Fe- and Al-P.Since the decrease of soil
pH is generally associated with the increase of Al- and
Fe-oxides, the common expectation is the increase of
NaOH-P with decrease in soil pH.
3.2. Pedogenic Ca-P
Citrate bicarbonate, and 2 consecutive citrate
extractions used in the sequential fractionation scheme
are proposed to remove pedogenic Ca-P (Ruiz et al.,
1997; Delgado et al., 2000). Ranges for CB-P, C1-P
and C2-P, were 41.5-261; 144-359 and 31-77 mg kg-
1
, respectively. Adhami et al. (2007) found that the
Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21
average content of the sum of CB-, C1- and C2-P in
16 highly calcareous soils of the south of Iran was
238, while in the present study it was 378 mg kg-1.
The content of pedogenic Ca-P is also higher than
those reported by Ruiz et al. (1997) in 10 slightly
acidic soils. Climate of Guilan province is sub-tropical
with an average rainfall of 1000 mm (Bahrami et al.,
2010) which has resulted in soils with lower pH, and
more Fe and Al oxides than soils from other parts of
the country. Thereupon, the general expectation was
a lower content of pedogenic Ca-P such as CB-, C1-
and C2-P (Cross and Schlesinger, 1995); the opposite
trend is probably caused by intensive application of
inorganic P fertilizers in the region.
It is likely that metastable phases (i.e. dicalcium ,
octacalcium, and tricalcium phosphate) and possibly
a small fraction of stable phase (hydroxyapatite)
were dissolved in CB reagent (Delgado et al., 2000).
Pedogenic Ca-P could be partly available to plants
(Samadi, 2006; Khanmirzaie et al., 2009). Delgado
et al. (2000) reported that part of Olsen P is originated
from CB-P. On the other hand citrate is common
reagent to evaluate P availability of fertilizers. The
high content of pedogenic Ca-P in the studied soils
indicates the high risks of adverse effects of P on both
agricultural production and environmental quality.
In the present study CB-P and C-P (sum of C1-
and C2-P) accounted for 12 and 21% of the sum P
fractions; respectively. Saavedra and Delgado (2005)
reported that the dominant P fractions in 17 soils from
Spain were CB-P and C-P which were accounted for
31% and 30% of the sum of P fractions; respectively.
Adhami et al. (2007) studying 16 calcareous soils of
Iran observed that CB-P and C-P constituted 9.4 % and
31% of the sum of P fractions.
Correlation of pedogenic Ca-P (CB, C1- and C2-P)
with soil properties showed no significant correlation
except for C1-P with silt and pH (Table 4). Multiple
regression analysis showed that pH and silt jointly could
predict 51 % of C1-P variation (Eq.1):
 Effect of soil properties on phophopus fraction in subtropical soils of Iran 15

Table 1. Some physico-chemical characteristics of the select soils.

a,
organic carbon;b, calcium carbonate equivalent; c, oxalate extractable Few; and d,
cation exchange capacity. Current land uses of
the soils are flooded rice; except for soil 11 which is under olive trees.

Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21

16 Adhami et al.
C1-P= 542 - 50.1 pH R2= 0.33 p<0.015
C1-P=358 - 44.3pH + 3.10 silt R2=0.51 p<0.007
Adhami et al. (2007) observed a positive correlation
between CB-P and silt fraction of the studied soils.
Delgado and Torrent (2000) believe that C-P
essentially correspond to sparingly pedogenic Ca-
P. Presumably, two consecutive citrate extractions,
should extract mainly pedogenic Ca-P compounds.
Delgado et al. (2000) explained that citrate in Ruiz’s
sequence essentially releases P related to Ca-phosphate
as a consequence of Ca complexation by citrate.
3.3. Forms of Reductant P
CAs-P ranged from w114 to 205 mg kg-1 (average
156 mg kg-1) and accounted for 14% of the sum of
P fractions. Citrate ascorbate extractable P can be
considered as mild reductant soluble P, and is assumed
to be P associated with poorly crystalline Fe oxides
(e.g. ferrihydrate) (Ruiz et al., 1997, Delgado et
al., 2000). The range of CBD-P was 36 to 262 mg
kg-1 with the average of 102 mg kg-1, and in average
constituted 9 percent of the sum of P fractions. Citrate-
bicarbonate-dithionite extractable P is part of soil-P
which is released with the effect of a strong reductant
(dithionite), generally is attributed to various forms
of P occluded in crystalline Fe oxides (Ruiz et al.,1997).
In most soils CAs-P was higher than CBD-P and in
average CAs-P was 1.5 fold of CBD-P. This indicates
that most of reductant soluble P in the studied soils
is “mild reductant P”. Ruiz et al. (1997) found that
CAs-P ranged from 13 to 133 mg kg-1 in 12 soils of
Europe and in most soils it accounted for more than
50% of reductant soluble P. Saavedra and Delgado
(2005) reported that the average amount of CAs-P
in 17 mediterranean soils was 11 mg kg-1. Although,
the results of chemical P fractionation could not be
Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21
(1) directly used to P dynamics in natural environments,
reductant soluble P (CAs-P and CBD-P) provides
an index of P that can be potentially released when
soil material are subjected to reducing condition in
(2)
natural environments. Thereupon, the high content
of CAs-P in the studied soils indicates that most of
the reductant soluble P could be easily reduced and
solubilized when subjected to reducing conditions,
i.e. in waterlogged rice fields or sediments of water
reservoirs.
In the present study, multiple regression analysis
showed that Feo could predict 59 percent of CAs-P
variation (Figure 1b); while introducing CCE as the
second variable increased R2 to 0.74 (Eq.3):
CA s-P =136 +0 .007F e o -2. 205C CER 2 =0.7 4p <0.0001
(3)
Although, citrate ascorbate is not a selective
extractant for P associated with Fe oxides in highly
calcareous soils (Ruiz et al., 1997; Adhami et al.,
2007); the results of the present study shows that
in acidic to slightly alkaline soils, citrate ascorbate
mainly extracts P associated with Feo. Relationship
between CAs-P and Feo is in accordance with the
assumption that CB followed by 2 citrate extractions
will remove all of the pedogenic Ca-P; therefore, the
use of citrate ascorbate should increase the P release as
a result of its reductant effect, and will not affect Ca-P
(Ruiz et al., 1997; and Delgado et al., 2000).
3.4. Lithogenic Ca-P
NaOAc-P ranged from 7.26 mg kg-1 to 99.6 mg
kg-1; and in average consisted 4 % of the sum of
inorganic P fractions. In slightly acidic to neutral
soils, NaOAc-P constituted 1 to 4 % of the sum
of P fractions while, in slightly alkaline soils
it constituted 1 to 9 % of the sum of P fractions.
Sodium acetate buffer pH 4 was introduced in
fractionation schemes to extract Ca phosphate
 Effect of soil properties on phophopus fraction in subtropical soils of Iran 17

Table 2. Sequence of the used fractination scheme

excluding lithogenic Ca-P (Jiang and Gu, 1989;

Ruttenberg, 1992; Barbanti et al., 2004; Ruiz
et al., 1997). Adhami et al. (2007) observed that NaOAc-P = 9.182 + 3.714 CCE R2=0.45 p<0.004 (4)
NaOAc-P in 16 calcareous soils from south of Iran
ranged from 8 to 76 mg kg-1 with an average of 36
mg kg-1. Ruiz et al. (1997) reported that NaOAc-P
NaOAc-P = -27.32 + 3.778 CCE + 1.116 Clay R2=0.70 p<0.001
ranged from 3 to 72 mg kg-1 in 12 noncalcareous and
(5)
calcareous soils of Europe. Relationship between
NaOAc-P and soil properties showed a significant
positive correlation with CCE, Clay and pH (Table
4). Multiple regression analysis showed that 70% of The relationships of NaOAc-P with CCE and
NaOAc-P variation could be predicted by CCE and clay are in accordance with the assumption that
Clay (Eq.5): NaOAc in the Ruiz et al. (1997) sequence is capable of

Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21

18 Adhami et al.
Table 3. Inorganic P fractions.
extracting Ca phosphate excluding lithogenic apatite.
Content of HCl-P ranged from 27 to 880 mg kg-1 with
an average of 419 mg kg-1 and in average constituted
37% of the sum of P fractions. Adhami et al. (2007)
reported that the average content of HCl-P in 16
calcareous soils of Iran was 125 mg kg-1. The content
of HCl-P is also more than those observed by Ruiz
et al. (1997) in 12 non-calcareous and calcareous
soils of Europe (4 to 129 mg kg-1). Saavedra and
Delgado (2005) found that the average content of
HCl-P in 17 agricultural soils of SW Spain was 14
mg kg-1. It is believed that phosphorus extracted
with a dilute acidic solution (e.g. HCl 0.25 M; or
H2SO4 0.25 M) is primary phosphorus minerals such
Journal of Soil Science and Plant Nutrition, 2013,13(1), 11-21
as hydroxylor flourapatite (Chang and Jackson, 1957;
Jiang and Gu, 1989; Adhami et al., 2007). Due to
higher weathering, general expectation was the
lower content of lithogenic Ca-P as the primary
phosphorus minerals (Cross and Schesinger,
1995) in the region than the soils of sourh of Iran,
the opposite trend is probably an effect of intensive
application of inorganic P fertilizer. It has been
reported heavily fertilized acid soils may contain
significant amounts of Ca phosphates (Lookman et
al., 1996), and hydroxyapatite has been found as a
product of the dissolution of superphosphates in
acid and slightly acidsoils (Kumar et al., 1994). The
least content of HCl-P was belong to soil 2 (pH=5.83)
 Effect of soil properties on phophopus fraction in subtropical soils of Iran 19

Table 4. Corelation coefficient between inorgancic P fractions and soil properties ( n = 17)

ns
, not significant; * and ** significant at p<0.05 and p<0.01; respectively.

4. Conclusion References

The results reported herein showed that the
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acidic to slightly alkaline soils of the north of Iran
was in the order NaOH-P (33 mg kg-1) < NaOAc-P
(39 mg kg-1) < C2-P (49 mg kg-1) < CBD-P (102 mg
kg-1) < CB-P (136 mg kg-1) < CAs-P (156 mg kg-1)
< C1-P (197 mg kg-1) < HCl-P (417 mg kg-1). The
studied soils showed a high inorganic P content,
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